In this renewal application, we seek to adapt our labeling strategies designed to minimize dehalogenation and maximize entrapment of radioactivity within tumor cells after receptor-mediated internalization of intact antibodies to nanobodies (Nbs) - the smallest naturally occurring antibody fragment. The small size (~15 kDa), nM-range affinity and low immunogenicity of these proteins make them a very attractive platform for the development of radiotheranostic agents. The goal of this proposal is to increase the clinical utility of labeled Nbs for cancer diagnosis and therapy by developing more effective strategies for labeling internalizing Nbs with radioiodine nuclides and 211At. Iodine-131 remains the most widely used radionuclide in clinical radioimmunotherapy; 211At emits ?-particles that have a greater cytotoxic effectiveness than conventional radiation and have a range in tissue of only a few cell diameters. Based on potential clinical impact and promising preliminary results, our strategy will be to focus on the development of HER2-targeted Nbs as molecular radiotherapeutics for HER2-positive breast carcinoma, particularly for patients with central nervous system (CNS) metastatic disease. Particularly in patients that respond to systemic treatments such as trastuzumab, there has been a rapid increase in the incidence of neoplastic meningitis (NM) and brain metastases (BM) to which these patients quickly succumb because of the lack of efficacy of these drugs against disease sites in the CNS. Unfortunately, these patients face a dim prognosis because current treatment options such as external beam radiotherapy are often dose limited because of CNS toxicity concerns. Our hypothesis is that by combining a HER2-targeted Nb construct with an optimized labeling method and radionuclide, this barrier will be overcome, thereby providing an effective strategy for the treatment of breast cancer patients with CNS metastases. In support of this approach, the results that we have obtained to date labeling the anti-HER2 Nb 5F7 using radio iodination methods originally designed for internalizing mAbs demonstrated enhanced retention of radioactivity in HER2-expressing tumor cells compared with conventional methods. Studies using N-succinimidyl 4-guanidinomethyl-3-[131I]iodobenzoate (SGMIB) were particularly encouraging, demonstrating a more than twofold tumor xenograft delivery advantage compared with and previously reported radionuclide/labeling method/Nb combination. Our Specific Aims are: 1) to label HER2 targeted Nb monomers and dimers with 131I and 211At using SGMIB and N-succinimidyl 3-[211At] astato-4- guanidinomethylbenzoate and to evaluate their potential as targeted radiotherapeutics: 2) to investigate strategies for improving Nb labeling; and 3) to evaluate the therapeutic efficacy of promising 131I- and 211At- labeled Nb conjugates in athymic rodents with subcutaneous, intracranial and neoplastic meningitis HER2- expressing xenografts.